in order to proliferate , cells must accurately transmit their chromosomes from one generation to the next . in eukaryotes ,
the chromosomes are confined to the nucleus , the perimeter of which is defined by the nuclear envelope .
the nuclear envelope is made of a double membrane ; the outer nuclear membrane is continuous with the endoplasmic reticulum ( er ) and contains many of the same proteins .
the inner nuclear membrane contains a distinct set of proteins , some of which interact with chromatin .
the outer and inner nuclear membranes are fused at sites of nuclear pore complexes ( npcs ) , macromolecular structures that allow selective passage of proteins , rna and other molecules to and from the nucleus . in most eukaryotes ,
underlying the inner nuclear membrane is a network of proteins , called the nuclear lamina , made of lamins and lamin - associated proteins .
this network provides rigidity to the nucleus and contributes to chromatin organization and other nuclear processes .
interestingly , neither plants nor fungi have a lamin - based nuclear lamina , although other structures may serve a similar purpose . of note
alterations in nuclear shape and size are characteristic of aging and certain disease states , such as various types of cancer .
thus , understanding how nuclear shape is regulated and identifying genes that contribute to nuclear morphology are of interest from both a medical and a basic biology standpoint .
the nuclear envelope not only serves as a diffusion barrier but also as a physical barrier separating the chromosomes from cytoplasmic structures . in most animal cells
these cytoplasmic structures include the centrosomes , which function as microtubule organizing centers that nucleate spindle microtubules necessary for chromosome segregation .
however , just before mitosis , the nuclear envelope stands between these microtubules and the chromosomes .
different cell types have adopted different strategies to facilitate the access of chromosomes by microtubules : at one end of the spectrum are cells of most metazoans , in which nuclear envelope components , including the two nuclear membranes , the npcs and the nuclear lamina with their associated proteins , disperse during each and every mitosis ( fig . 
this type of mitosis is called  open mitosis ,  and it necessitates the reassembly of the nuclear envelope around a full complement of chromosomes in each of the daughter cells once chromosome segregation is completed .
parenthetically , the mechanism that ensures the formation of only a single nucleus at the end mitosis , as opposed to several nuclei each encompassing a subset of chromosomes , is not known . at the other end of the spectrum
are certain fungi in which the centrosome equivalents , called spindle pole bodies , are either permanently embedded in the nuclear envelope ( such as in budding yeast ) or are embedded in the nuclear envelope prior to mitosis ( such as in fission yeast ) .
these cells undergo what is called  closed mitosis ,  which occurs without nuclear envelope breakdown , as spindle microtubules that assemble within the nucleus can readily access the chromosomes
. however , during closed mitosis , the nucleus must elongate in a manner that is coordinated with chromosome movement as chromosome segregation takes place ( fig . 
the chromatin ( blue ) is contained within the nuclear envelope ( light green ) .
as cells enter mitosis , the nuclear envelope disassembles , allowing spindle microtubules ( purple lines ) nucleated by centrosomes ( purple spheres ) to align the chromosomes on the metaphase plate .
( b ) closed mitosis . shown is mitosis as it occurs in s. cerevisiae .
the spindle pole body ( purple ) is embedded in the nuclear envelope throughout the cell cycle .
after spindle pole body duplication , an intra - nuclear spindle is formed ( s. cerevisiae chromosomes do not condense enough to visualize individual chromosomes or a metaphase plate ) . during anaphase , the nucleus elongates and the nuclear envelope expands as the sister chromatids move away from each other .
( c ) functionally open , structurally closed mitosis . a term defined by sazer to indicate a breakdown in the diffusion barrier while maintaining an intact nuclear envelope . shown is mitosis as it occurs in aspergillus nidulans . as in s. cerevisiae , during interphase the spindle pole body is embedded in the nuclear envelope .
as cells enter mitosis the nuclear pores ( dark green ) partially disassemble , significantly reducing the diffusion barrier between the nucleus and cytoplasm . as in closed mitosis ,
the bulge in the center of the elongated nucleus during anaphase contains the nucleolus , which is left behind during chromosome segregation .
schizosaccharomyces japonicus cells form an intra - nuclear spindle as in cells undergoing closed or partially open mitosis .
however , during anaphase , the nuclear envelope ruptures as the nucleus elongates without nuclear envelope expansion . the historic definition of open vs. closed mitosis was based on cytology : during open mitosis the nuclear envelope disappears ,  opening  the nucleus and exposing its content to the cytoplasm , while in closed mitosis the nuclear envelope remains intact . from a functional standpoint , the presence or absence of a nuclear envelope affects at least two processes : the free diffusion of molecules between the cytoplasm and the nucleus ( which occurs during open , but not closed , mitosis ) and the presence of a physical barrier between the chromosomes and cytoplasmic structures ( which exists in closed , but not open , mitosis ) . over the years , mitotic divisions that fall somewhere in between open and closed have been described , warranting a brief discussion on how these forms of mitosis should be defined . in aspergillus nidulans , which was originally defined as undergoing closed mitosis , npcs
partially disassemble as cells enter mitosis , disrupting the diffusion barrier between the nucleus and the cytoplasm , while maintaining a physical barrier between the chromosomes and cytoplasmic structures ( fig . 
a breakdown in the diffusion barrier also occurs during a brief period in anaphase of meiosis ii of the fission yeast schisosaccharomyces pombe . unlike the situation in a. nidulans
, however , the change in permeability in s. pombe meiosis ii occurs at only a short window of nuclear division and is not accompanied by changes in either npc composition or nuclear membrane integrity .
this led sazer to define this type of division as  functionally open but structurally closed 
, a term that can also be applied to a. nidulans mitosis . other types of mitosis involve a partial opening in the nuclear membrane itself . in schisosaccharomyces japonicas ,
the nuclear envelope does not expand during mitosis , nor does it disassemble . as a result , the nuclear envelope ruptures as the intra - nuclear spindle elongates ( fig . 
interestingly , rupturing is not a result of forces exerted by the spindle , but rather a programed cell cycle event , the regulation and mechanism of which remain to be discovered . in the early embryonic divisions of the nematode caenorhabditis
elegans the nuclear envelope is breached in pro - metaphase , allowing diffusion of cytoplasmic proteins into the nuclear space .
a similar phenomenon is seen during drosophila syncytial divisions , where the nuclear envelope displays a partial disassembly of nuclear pore complexes and fenestrations near the centrosomes , thereby allowing microtubules to access the chromosomes .
live cell imaging suggests that just as in closed mitosis , the nuclear envelope during drosophila syncytial divisions expands as the spindle elongates . in these three cases , however , mitosis is not structurally closed , as nuclear envelope integrity is breached and cytoplasmic structures , such as microtubules , can enter the nucleus . consequently , the term  functionally open but structurally closed  may not fully apply when describing these types of mitosis , and several studies have previously referred to these mitoses as  semi - open mitosis . 
we propose to use  partially open mitosis  as term that encompasses all forms of mitosis that are neither fully closed nor fully open , but that exhibit a change in the diffusion barrier at some point during mitosis that may or may not be accompanied by visible structural change to the nuclear envelope . among unicellular eukaryotes
there are many variations of nuclear envelope behavior during mitosis ( for more examples see refs .
16 and 17 ) , and undoubtedly additional forms of mitosis still remain to be discovered .
it is likely that the various types of mitosis have evolved to adapt to a particular environment , although it is currently not known what kinds of selective pressure shaped the different modes of mitosis .
a possible driving force is the position of the centrosome , namely in the cytoplasm vs. embedded in the nuclear envelope , which likely coevolved with nuclear envelope behavior .
in addition , the evolution of mitosis could have been affected by the need to eliminate rate - limiting diffusion barriers between the nucleus and the cytoplasm as occurs during open and partially open mitosis , but is either not a problem or is somehow overcome in closed mitosis .
it is conceivable that , during syncytial divisions , partially open mitosis evolved to overcome the diffusion barrier and microtubule accessibility problems , while protecting chromosomes from aberrant attachments to microtubules from neighboring nuclei .
the evolution of different forms of mitosis remains an unresolved , yet fascinating , question .
closed mitosis appears at first glance to be an economical solution to the issue of microtubule - chromosome accessibility : nuclear envelope breakdown is circumvented , the chromosomes do not get  entangled  with cytoplasmic structures , and cells do not have to reassemble the nuclear envelope at the end of mitosis , risking leaving one or more chromosomes outside the nuclear perimeter .
chief among them is how the nuclear envelope expands . in particular , how is the timing of nuclear expansion determined ?
is it a consequence of spindle elongation or is it an independent process regulated by the cell cycle machinery ? and how are nuclear envelope components added ?
finally , what happens to nuclear envelope expansion if cells are delayed in mitosis , for example due to checkpoint activation ?
an insight into these questions came from a study by witkin et al . on the shape of the budding yeast nucleus . during normal growth ,
the budding yeast nucleus is round , except during mitosis when it elongates through the bud neck to accommodate chromosome segregation ( fig . 
witkin et al . screened for genes that when deleted lead to alterations in nuclear shape , aiming to identify genes that affect nuclear envelope dynamics .
the authors used a collection of roughly 5000 budding yeast deletion mutants ( each deleted for a single non - essential gene ) , into which they introduced a nuclear gfp marker and a cytoplasmic rfp marker .
this strategy then allowed them to screen visually for mutants that displayed abnormal nuclear morphology .
one class of nuclear abnormalities was of nuclei that exhibited extensions , referred to as  flares 
. further examination of these mutants revealed that this altered nuclear phenotype was not due to a direct involvement of the deleted genes in nuclear morphology , but rather due to the activation of a checkpoint pathway ( e.g. , the dna damage checkpoint or the spindle assembly checkpoint ) that induced a mitotic delay .
in other words , the mutations isolated in the screen likely increased the formation of intracellular damage , which , in turn , led to a mitotic delay through checkpoint activation . indeed , other conditions that induced a mitotic delay ( e.g. , depolymerizing microtubules with nocodazole ; inhibiting the anaphase promoting complex ) also led to the formation of a nuclear flare .
interestingly , the nuclear flare formed due to a mitotic delay was confined to the nuclear envelope region adjacent to the nucleolus ( fig .  2b , and see below ) , which normally forms a crescent - shaped structure at the nuclear periphery ( fig . 
are images taken 20 min apart of budding yeast cells expressing a gfp - tagged nucleoplasmic marker ( pus1 ) and an rfp - tagged nucleolar marker ( nsr1 ) .
importantly , the change in nuclear morphology was specific to a cell cycle delay in mitosis ; witkin et al . found that nuclei of cells arrested in s phase or g2 were mostly round . moreover , the formation of the nuclear flare in cells treated with nocodazole began at the same time as nuclear elongation in untreated cells and was dependent on phospholipid synthesis .
however , phospholipid synthesis alone was not sufficient to induce flares , as g2 arrested cells accumulated just as much phospholipid as mitotic arrested cells . taken together , these results suggest that nuclear expansion during closed mitosis of budding yeast is independent of spindle elongation , but rather occurs in response to a cell cycle cue that signals mitotic entry .
first , what regulates nuclear envelope expansion ? as noted above , the process is independent of spindle elongation , much like nuclear envelope rupture in s. japonicus .
thus , in these yeasts there are likely targets of the cell cycle machinery that affect nuclear envelope dynamics during mitosis
the synthesis of phospholipids , the main components of the nuclear membrane , is required for nuclear envelope expansion , as shown previously for both budding and fission yeast .
moreover , at least one enzyme that negatively regulates membrane expansion , pah1 , is regulated by cdk1 phosphorylation ( ref .
the observation that a nuclear flare forms when checkpoint pathways are activated suggests that phospholipid synthesis is not subjected to checkpoint regulation . however , phospholipid synthesis is unlikely to be the only cell cycle target required for nuclear expansion , because g2-arrested cells accumulate just as much phospholipid as mitotic - arrested cells , yet they do n't form a flare ( ref .
18 ; note , however , that it is currently not known whether nuclear envelope of g2 cells also expands , but without forming a flare ) .
it is tempting to speculate that the cell cycle target may be a protein or a process that is responsible for allocating membrane addition specifically to the nuclear envelope , either by localized phospholipid synthesis or by drawing membrane from the er preferentially during mitosis .
spindle elongation may also play a role in nuclear envelope expansion : although the surface area of the nucleus increases during a mitotic arrest in the absence of a spindle ( i.e. , during an arrest induced by nocodazole ) , it is possible that spindle elongation contributes to the rate , and perhaps magnitude , of nuclear envelope expansion .
observed that nuclear envelope expansion during a mitotic arrest occurs in a curious fashion : rather than expanding isometrically , thereby generating a larger sphere , the flare occurs at the nuclear envelope that is adjacent to the nucleolus ( fig . 
whether this occurs in other organisms that undergo closed mitosis is currently not known , as cell cycle analyses typically use dna staining , rather than a nuclear envelope marker , to follow cell cycle progression .
nonetheless , the flare induced by a mitotic arrest is similar to the nuclear expansion observed when the pah1 pathway is misregulated .
pah1 converts phosphatidic acid to diacylglycerol . in the absence of pah1 ( or in the absence of its activators , the nem1 and spo7 phosphatase complex ) the overall levels of certain phospholipids increase ,
the er loses its tubular shape in favor of membrane sheets , and the nucleus forms a flare at the nuclear envelope adjacent to the nucleolus .
why , then , in both pah1 pathway mutants and in a mitotic arrest is the expansion of the nuclear envelope specific to the region adjacent to the nucleolus ?
campbell et al . showed that in a spo7 mutant , if the nucleolus is reduced to a tiny sphere the flare still forms , with the diminished nucleolus often at the base of the flare .
whether the nucleolus , or its remnant , affects the adjacent nuclear envelope remains unknown .
alternatively , there may be a structure that prevents nuclear envelope expansion around the entire nuclear envelope except in the region adjacent to the nucleolus .
there are indeed budding yeast nuclear membrane proteins that are excluded from the nucleolus ; none of the ones tested had properties consistent with an inhibitor of membrane expansion ( our lab , unpublished ) , but other such proteins may still exist .
since the nuclear envelope is continuous with the er , it is quite possible that phospholipids are synthesized at the nuclear envelope itself .
if this were the case , it is conceivable that the nuclear envelope adjacent to the nucleolus would have a higher capacity for phospholipid synthesis , or that membrane is added throughout the nuclear envelope , but , as mentioned above , there may be a mechanism that resists expansion everywhere except adjacent to the nucleolus .
alternatively , the membrane could come from the er , and if this were the case then the cell would have a mechanism for allocating membrane from the er to the nuclear envelope specifically during mitosis .
it is currently not known whether the er expands during a mitotic arrest , and if so , at what rate .
thus , the mechanisms driving nuclear envelope dynamics , and in particular nuclear envelope expansion , remain to be uncovered .
the study by witkin et al . highlights the dynamic nature of the nuclear envelope during the cell cycle . the observations in this study support a model whereby
the nuclear envelope adjacent to the nucleolus is the preferential site of expansion during a mitotic delay .
it is interesting that phospholipid synthesis is not under checkpoint control ; consequently , a cell delayed in mitosis needs to somehow deal with increased nuclear envelope surface area in the absence of chromosome segregation .
we can envision at least three possibilities : first , sequestering the added nuclear envelope to the region adjacent to the nucleolus could preserve inter - chromosomal interactions that would otherwise be disrupted were the nucleus to expand isometrically .
second , yeast cells are known to maintain a constant nuclear to cell volume ratio by a yet unknown mechanism .
it is possible that by confining the extra nuclear envelope to a flare , rather than distributing it throughout the entire nuclear surface , the cell is better able to regulate its nuclear volume .
finally , the nuclear envelope adjacent to the nucleolus may be the region of initial nuclear envelope expansion even under normal growth conditions , but in the absence of spindle elongation this expansion is not distributed throughout the entire nuclear surface .
regardless of the mechanism , the appearance of a nuclear flare suggests that the budding yeast nuclear envelope has domains of distinct properties . how these domains differ in their ability to expand is not known .
isolation of mutants that fail to produce a flare during a mitotic arrest will likely shed light on the mechanism that designates distinct nuclear envelope domains and on the function of flare formation . finally , can observations in closed mitosis illuminate processes related to the nuclear envelope in cells that undergo open mitosis ? as in closed mitosis , the nuclear envelope of metazoans must also expand , not during mitosis but during interphase , immediately following nuclear envelope reassembly .
much like in yeast , the source of membrane , and both the temporal and spatial regulation of this process , are unknown .
moreover , in certain cancer cells nuclei become larger and multi - lobed , and during aging the nuclear envelope appears 
if so , do changes in the nuclear envelope have consequences for cell function ? despite the different modes of mitosis in budding yeast and mammalian cells , cell cycle processes have often proved to be based on conserved proteins .
thus , studies on nuclear envelope dynamics in budding yeast will likely shed light on processes related to nuclear envelope formation , tumorigenesis and aging in higher eukaryotes .
 in eukaryotes , chromosomes are encased by a dynamic nuclear envelope . in contrast to metazoans , where the nuclear envelope disassembles during mitosis , many fungi including budding yeast undergo  closed mitosis ,  where the nuclear envelope remains intact throughout the cell cycle . 
 consequently , during closed mitosis the nuclear envelope must expand to accommodate chromosome segregation to the two daughter cells . 
 a recent study by witkin et al . in budding yeast showed that if progression through mitosis is delayed , for example due to checkpoint activation , the nuclear envelope continues to expand despite the block to chromosome segregation . 
 moreover , this expansion occurs at a specific region of the nuclear envelope- adjacent to the nucleolus- forming an extension referred to as a  flare .  
 these observations raise questions regarding the regulation of nuclear envelope expansion both in budding yeast and in higher eukaryotes , the mechanisms confining mitotic nuclear envelope expansion to a particular region and the possible consequences of failing to regulate nuclear envelope expansion during the cell cycle . 
The Nuclear Envelope during Mitosis: Anything but Static
Closed Mitosis: A Strategy that Generates a Complication
Regulation of Nuclear Envelope Expansion
The Expanding Mitotic Nuclear Envelope Uncovers Discrete Nuclear Envelope Domains
Conclusions and Open Questions