microdissection , genetic , and inhibitor experiments have been used to define the parts of the spindle that are required for cleavage furrow induction .
chromosomes have been shown to be dispensable for cytokinesis ( rappaport , 1996 ; zhang and nicklas , 1996 ; bucciarelli et al . , 2003 ; dekens et al . , 2003 ) .
likewise , centrosomes can be ablated or genetically disrupted without preventing cytokinesis ( khodjakov and rieder , 2001 ; megraw et al . , 2001 ) .
though chromosomes and centrosomes are dispensable , they may influence the process when they are present ( piel et al . , 2001 ) .
in addition to chromosomes and centrosomes , the spindle contains a large array of microtubules .
microtubule depolymerization during metaphase or very early anaphase prevents cleavage furrow formation , indicating that microtubules are essential ( hamaguchi , 1975 ) . however , furrow formation can occur if the mitotic spindle is depolymerized later in anaphase , but before ingression has begun ( hamaguchi , 1975 ) .
thus , mitotic spindle microtubules are required to induce furrow formation , but they are not , per se , required for ingression . further insight into the mechanism of cleavage furrow induction has come from experiments in which cells , usually embryos , are physically manipulated and their potential to cleave assessed .
these perturbations include alteration of the position of the spindle with respect to the cell cortex , cell shape deformation , and removal of parts of the spindle .
for example , the classic  torus experiment  in which two spindles in a common cytoplasm induce an additional furrow indicates that opposing asters are sufficient to induce a furrow ( rappaport , 1961 ) .
additionally , repositioning of the spindle during anaphase results in multiple cleavage furrows whose positions are dictated by the spindle ( rappaport , 1985 ) .
results of numerous experiments of this type have led to the astral stimulation model ( fig .
this concept assumes that astral microtubules provide a cleavage stimulus , which , for example , could be a factor that is transported along astral microtubules .
this model proposes that because the equatorial cortex is influenced by astral microtubules from two poles , the strength of this stimulus would be highest at the cell equator . with some assumptions concerning the nature of the signal , its mode of delivery , and the distribution of microtubules
, computer modeling indicates that a cleavage stimulus could reach a maximum at the equatorial region ( devore et al . , 1989 ;
, asserts that astral rays ( i.e. , microtubules ) cause a reduction of cortical contractility in a density - dependent manner . according to this model , the density of astral rays is higher near the poles than at the equator , assuming spherically symmetric asters in spherical cells .
this would cause the polar regions to be less contractile than the equator , and this difference in contractility would induce equatorial furrowing ( fig . 1 b ; wolpert , 1960 ) .
quantitative modeling confirmed that this model could , in principle , allow furrow formation , but indicated that a positive feedback loop during contractility would be required to allow complete ingression ( white and borisy , 1983 ; yoshigaki , 1999 ) .
these two models come to opposite conclusions regarding the role of astral microtubules because they differ in their underlying assumptions about the distribution of microtubules , their lengths , and the way in which they interact with the cell cortex .
in addition , it is now apparent that activities exist that bundle microtubules from opposing asters and generate a structure that is called the central spindle ( also known as the spindle midzone ) .
the evolutionarily conserved centralspindlin complex containing a kinesin - like protein mklp1 and a rho family gap , hscyk-4/mgcracgap ( mishima et al . ,
centralspindlin is directly involved in central spindle assembly because it localizes to the central spindle and has microtubule - bundling activity ( mishima et al . ,
another important factor in central spindle assembly is the microtubule - binding protein prc1 ( mollinari et al . , 2002 ) .
because there is evidence that antiparallel microtubule bundles can regulate furrowing ( see below ) , some of the micromanipulation experiments that have led to the astral stimulation and relaxation models may need to be reinterpreted .
indeed , observations in drosophila provide compelling evidence that astral microtubules may not be critical for furrow formation and that the central spindle is necessary and sufficient to induce furrow formation ( fig
in particular , cells deficient in the kinesin - like protein pavarotti ( the orthologue of hs mklp1/ce zen-4 ) fail to form a central spindle , have rather normal appearing astral microtubules , and do not form cleavage furrows ( adams et al .
conversely , asterless mutants , which lack most astral microtubules but retain a central spindle , are still capable of forming cleavage furrows ( bonaccorsi et al . , 1998 ) .
these data fit neither the astral stimulation nor the astral relaxation model , and suggest that the central spindle is responsible for furrow induction .
additional evidence supports the notion that the central spindle is involved in furrow formation . in cultured rat cells ,
if a small perforation is created adjacent to the central spindle , furrow formation occurs on the side of the perforation adjacent to the central spindle and not at the cortical site where furrow formation would have occurred in an unmanipulated cell ( cao and wang , 1996 ) .
furthermore , grasshopper spermatocytes have been manipulated to simultaneously remove centrosomes and chromosomes , and the remaining microtubules self - organize into bundles that resemble the central spindle and appear to induce furrow formation ( alsop and zhang , 2003 ) .
these results , combined with the fact that many key regulators of mitotic events localize to the central spindle , have lead to the proposal that central spindle microtubules ( or more generally , antiparallel microtubule bundles ) are a principle regulator of furrow formation . however , there is also compelling evidence that the central spindle is dispensable for cleavage furrow formation . in caenorhabditis elegans embryos ,
cleavage furrows form and constrict , but they fail to complete cytokinesis ( powers et al . , 1998 ; raich et al .
, 1998 ; jantsch - plunger et al . , 2000 ) . the dramatically different requirement for the central spindle in furrow formation in drosophila and c. elegans could result from differences in cell size in these systems .
indeed , some variation has been reported in the localization of critical factors that regulate cytokinesis .
for example , in drosophila , in addition to the central spindle localized pool of pavarotti , there is also a cortically localized pool that is not detected in other organisms ( sellitto and kuriyama , 1988 ; adams et al . , 1998 ; powers et al .
conversely , in mammalian cells , ect2 ( a gef for rhoa ) is readily detected in association with both the cell cortex and the spindle , but its orthologue in drosophila is primarily associated with the cell cortex ( prokopenko et al . , 1999 ;
however , recent results suggest that neither cell size nor lack of conservation underlies the variable degree to which the central spindle controls furrow formation , and indicate that this process is controlled by two parallel pathways . in c. elegans embryos , the central spindle is not generally essential for furrow formation .
however , if the extent of spindle elongation during anaphase is reduced by one of several genetic perturbations , the central spindle becomes essential ( dechant and glotzer , 2003 ) .
in addition , although furrow formation can occur in the absence of the central spindle , initiation of cytokinesis is slightly delayed under these circumstances .
thus , perhaps different cell types use both astral microtubules and the central spindle for furrow formation , albeit to varying degrees .
indeed , there is evidence for plasticity in the induction of cleavage furrows in mammalian cells .
microsurgical experiments indicate that the central spindle has furrow - inducing activity , yet cells depleted for key central spindle components , such as mklp1 or prc1 , still form furrows ( cao and wang , 1996 ; matuliene and kuriyama , 2002 ; mollinari et al . ,
given that both the central spindle and astral microtubules can contribute to induction of cleavage furrows , at least under some circumstances , proteins that localize to these structures are potential clues to the mechanism of furrow induction .
, these factors could regulate furrow formation in two ways : they could be positive inducers of furrow formation , or they could inhibit a negative regulator of furrow formation .
there are several factors that concentrate on the central spindle that have been suggested to be inducers of cleavage furrow formation .
one candidate is the abi complex consisting of aurora b , incenp , and survivin / bir-1 ( adams et al . , 2000 ;
, 2001 ; bolton et al . , 2002 ; cheeseman et al . , 2002 ; honda et al . , 2003 ; romano et al . ,
this complex contains a fourth protein , csc-1 ( romano et al . , 2003 ) .
in mammalian cells , incenp first localizes to chromosomes during prometaphase , then it concentrates on centromeres during metaphase , and then , upon anaphase onset , it localizes to both the central spindle and , interestingly , the overlying cell cortex ( cooke et al . , 1987 ) .
both astral microtubules and the central spindle contribute to cortical localization of aurora b ( murata - hori and wang , 2002 ) , presumably due to interactions with incenp and survivin , whose sole function appears to be to activate and localize aurora b. interestingly , aurora b localizes to the central spindle in cells that lack chromosomes ( bucciarelli et al . , 2003 ) , indicating that these subcellular targeting events are independent .
the cortical localization of the abi complex precedes the early stages of cytokinesis ( eckley et al . , 1997 ) .
although this localization of the abi complex suggests that it may direct cleavage furrow formation , cells deficient in aurora b ( due to mutation , rnai - mediated depletion , or chemical inhibition ) are competent to form cleavage furrows ( schumacher et al .
, 1998 ; fraser et al . , 1999 ; kaitna et al . , 2000 ; hauf et al . ,
a second potential activator of cleavage furrow formation that could link the central spindle to cleavage furrow formation is the rhogef , pebble .
pebble was recently shown to associate with drosophila centralspindlin ( somers and saint , 2003 ) .
pebble ( hs ect2/ce let-21 ) is essential for furrow formation , presumably because it is the critical activator of rhoa in cytokinesis ( prokopenko et al .
two - hybrid analysis indicates that the nh2 terminus of pebble binds to the nh2-terminal region of the fly orthologue of cyk-4 , racgap50c .
concentration of centralspindlin in the spindle midzone could thereby recruit pebble and induce the local activation of rhoa , followed by actin polymerization and cleavage furrow formation .
if this were the case , then cells defective in central spindle formation would also be expected to be defective in furrow formation .
although coupling of these two processes is observed in drosophila , this is not the case in c. elegans embryos or in mammalian cells .
moreover , overexpression of the nh2-terminal domain of the pebble orthologue , ect2 , causes a late defect in cytokinesis ( tatsumoto et al . , 1999 ) ,
not the early defect expected if the association of pebble with centralspindlin was essential for spatial regulation of pebble function .
thus , although pebble is critical for furrow formation , its association with the central spindle does not appear to be critical in all species .
the association of pebble with centralspindlin might promote the continued ingression of the cleavage furrow by maintaining rhoa in an active state .
it will certainly be interesting to understand the interplay between the rhogap and the rhogef in this unusual protein complex .
an alternative way to regulate furrow formation is through local inhibition of a negative regulator .
experiments in mammalian cells and c. elegans embryos suggest that during cytokinesis , microtubules inhibit the contractility of the cell cortex . when microtubules are induced to be unusually short by prolonged activation of the katanin microtubule - severing complex ,
similarly , mammalian cells forced to exit mitosis in the absence of microtubules undergo vigorous unorganized contractions ( canman et al . , 2000 ) .
given that there is compelling evidence that microtubules control furrow formation , it is conceivable that modulation of their distribution or properties could control furrow formation . in principle , bundling of microtubules to form the central spindle could quantitatively and/or qualitatively regulate the inhibitory effects of microtubules .
thus , a second mechanism by which the central spindle could promote furrow formation is by inhibiting this negative regulation .
experimental evidence that furrow positioning may result from local relief from microtubule - dependent inhibition of furrow formation .
( a ) stabilization of the microtubule - severing complex katanin leads to microtubule shortening and ectopic furrowing .
( b ) either central spindle assembly or spindle elongation are sufficient to induce furrow formation , but when both pathways are inhibited , no furrow formation occurs .
( c ) in mammalian cells , a subset of the microtubules in the vicinity of the furrow are less dynamic ( purple ) than elsewhere in the cell .
spindle elongation and central spindle assembly act together to create a local minimum of microtubule density at a position in the cell equator equidistant from the two spindle poles ( dechant and glotzer , 2003 ) .
importantly , these two processes also act in parallel to promote furrow formation ( fig .
these observations are consistent with a model in which the position of the cleavage furrow in the equatorial region is determined by a site where the inhibitory effect of microtubules reaches a local minimum ( fig .
central spindle assembly may not only affect the spatial organization of microtubules  it could also alter the capacity of microtubules to inhibit contractility by changing the properties of the microtubules .
for example , inhibition of cortical contractility could rely on the dynamics of microtubules or microtubule - associated proteins .
binding of factors to the ends of the microtubules could alter their properties or dynamics .
indeed , early work in mammalian cells indicated that midzone microtubules are more stable than elsewhere in the cell ( saxton and mcintosh , 1987 ) , and more recent observations confirm and extend these findings .
in particular , before furrow ingression , a subset of microtubules in the vicinity of the presumptive furrow are significantly more stable than microtubules near the cell poles ( fig .
it is not yet known if the stabilization of microtubules in the equatorial region is mediated by the centralspindlin complex , but this seems likely because it is present there , and overexpression of the kinesin subunit of centralspindlin ( mklp1/pavarotti ) induces hyperstabilization of microtubules ( minestrini et al . , 2002 ) .
thus , binding of centralspindlin to microtubules could induce central spindle assembly and simultaneously prevent the microtubule - dependent inhibition of furrow formation .
the rhoa exchange factor , dm pebble ( hs ect2 , ce let-21 ) is one of the most upstream molecules in this pathway and is a candidate for regulation by microtubules .
however , little is known about how this critical exchange factor is regulated , except that its activity requires phosphorylation ( tatsumoto et al . , 1999 ) . additionally ,
although rhoa and some of its effectors concentrate at the cleavage furrow , there is no direct evidence for local differences in rhoa activity early in cytokinesis .
recently , a fret - based approach to observe active rhoa during cytokinesis was reported .
these probes did not reveal detectable amounts of active rhoa during early cytokinesis , but active rhoa did appear late in cytokinesis ( yoshizaki et al . , 2003 ) . because rhoa activity is required for the initial stages of furrow formation , it is possible that low levels of active rhoa drive furrow formation , and these levels of rhoa were below the detection limit of these probes .
second generation probes specific for active forms of rhoa and other molecules essential for cytokinesis may provide further insight into this important question .
however , given that there are several precedents for local activation of gtpase signaling complexes , a reasonable working model is that a local increase in rhoa signaling induces furrow formation .
if so , furrow positioning could simply be explained if astral microtubules inhibit rhoa activation , thereby inhibiting furrow formation at ectopic sites , and central spindle assembly and spindle elongation conspire to provide local relief from these inhibitory effects and allow rhoa activation at the equatorial region .
interestingly , microtubule depolymerization in interphase cells causes activation of rhoa - gtp ( ren et al . , 1999 ) , implying that microtubules can , directly or indirectly , inhibit rhoa .
moreover , a particular rhogef , gef - h1 , is inhibited by microtubule - mediated sequestration ( krendel et al . , 2002 ) , illustrating one such mechanism .
further analysis of the biochemical events that occur during furrow initiation is absolutely essential , with particular attention paid to how these events might be regulated by microtubules .
at this juncture , furrow positioning does not appear to be solely due to induction by astral microtubules or the central spindle , but rather , both components contribute .
cleavage furrow induction through local relief from the inhibitory effects of microtubules is an appealing model because it explains how two pathways , spindle elongation and central spindle assembly , could control furrow formation through a common molecular mechanism .
in addition , it accounts for the fact that the central spindle has a positive ( though nonessential ) role in furrow formation .
it also has predictive value in that local depolymerization of microtubules should induce cleavage furrows .
in particular , it is tempting to speculate that the signals discussed here are important for patterning of the cortex in response to a local inhomogeneity in the distribution of microtubules .
subsequent reactions may be required to refine this positional information and to amplify the signal that directs assembly of the contractile ring .
alternatively , contractile ring assembly might be a cooperative process that is self - refining and amplifying .
 to complete the cell cycle , the cleavage furrow draws the plasma membrane toward the cell center , pinching the cytoplasm into two lobes that are subsequently separated into two cells . 
 the position of the cleavage furrow is induced by the mitotic spindle during early anaphase . 
 although the mechanism of cleavage furrow positioning is not understood at a molecular level , recent results suggest that it might be mediated by local relief from the inhibitory effects of microtubules . 