This represents a novel role for dynein during mammalian spindle assembly. Because centrosome separation in prophase requires dynein, presumably anchored to the nuclear envelope acting on astral MTs, as well as Eg5, acting on antiparallel MTs [2,23], we propose that as mitosis progresses and centrosomes separate, dynein becomes recruited to newly forming regions of antiparallel overlap where it can antagonize the activity of Eg5 and limit or stabilize centrosome separation, so as to prevent anaphaselike prometaphase [24]. capitalize on this relationship, we utilized a nocodazole washout assay to mimic spindle assembly. We found that Eg5 inhibition led to either monopolar or bipolar spindle formation, depending on whether centrosomes were initially separated less than or greater than 5.5m, respectively. Mathematical modeling predicted this same spindle bistability in the absence of functional Eg5, and required dynein acting BAPTA/AM on antiparallel MTs to do so. Our results suggest that dynein functionally coordinates with GADD45B Eg5 by crosslinking and sliding antiparallel MTs, a novel role for dynein within the framework of spindle assembly. == Results and Discussion == == Eg5/Dynein Antagonism in LLC-Pk1 Cells == Before exploring the functional coordination of Eg5 and dynein, we first confirmed the antagonistic nature of these motors in LLC-Pk1 cells, as well as the relevance of this antagonism to spindle bipolarity [3]. To accomplish this, metaphase cells expressing GFP-tubulin BAPTA/AM (LLC-Pk1 [7]) were treated with monastrol to inhibit Eg5 [8] or injected with p150-CC1 to inhibit dynein [9]. Immediately following monastrol treatment, bipolar spindles shortened by ~30% (Figures S1A and S1C), but complete collapse into monopoles was not observed. Conversely, spindles lengthened by ~30% after dynein inhibition (Figures S1B and S1C). This spindle elongation was not a consequence of centrosome dissociation from spindle poles or mislocalization of Kif2a (unpublished data). Together, spindle shortening and lengthening following Eg5 and dynein inhibition, respectively, confirm the presence of an antagonistic relationship between these two motors in LLC-Pk1 cells. We next monitored the response of Eg5-inhibited monopolar spindles to inhibition of dynein. LLC-Pk1 cells were treated with monastrol prior to nuclear envelope breakdown, and mitotic cells containing monopolar microtubule (MT) arrays were then injected with p150-CC1. In ~50% of such cells (13 of 30 cells), monopolar spindles reorganized into bipolar spindles (Figure S1DandMovie S1), defined here and subsequently as fusiform MT arrays with the majority of chromosomes aligned between two distinct poles separated by a minimum of 5m. Bipolar spindles that formed following inhibition of Eg5 and dynein were morphologically and functionally equivalent to controls (Figure S2). Spontaneous bipolarization of monastrol-induced monopolar spindles was never observed and injection of control antibodies left monopolar arrays unaltered (unpublished data), demonstrating the specificity of bipolarization to dynein inhibition. The formation of bipolar spindles following inhibition of Eg5 and dynein demonstrates that an additional force drives pole separation. This force could be generated by MT polymerization [10] and/or additional plus end-directed motors, such as Xklp2 [11]. Residual Eg5 activity, however, is unlikely to contribute to pole separation, given the efficacy of motor inhibition by monastrol [8]. In contrast, incomplete inhibition of dynein following injection of p150-CC1, which interferes with the dynein/dynactin interaction but not dyneins ATPase activity, could account for the observation that not all monopolar spindles were rescued. Finally, the geometry of MTs and chromosomes in co-inhibited cells may influence the generation of pushing forces that restore spindle bipolarity. In summary, the observation BAPTA/AM that dynein inhibition can rescue the monastrol-mediated monopolar phenotype demonstrates that an antagonistic balance between Eg5 and dynein contributes to the establishment of spindle bipolarity. Based on the evidence that Eg5 can slide apart overlapping MTs [46], and its antagonistic relationship with dynein [3], we hypothesize that dynein functions at regions of antiparallel overlap, where it crosslinks and slides antiparallel MTs in opposition to Eg5. == The Nocodazole Washout Assay as a Means to Study Eg5/Dynein Antagonism == If our hypothesis is valid, then dynein would likely be responsible for monopolar spindle formation in the presence of monastrol, as it would generate an inadequately opposed inward force. Because antiparallel overlap decreases as the distance between centrosomes increases (Figure S3A), and because the magnitude of our postulated dynein-mediated force would depend on the amount of antiparallel overlap, spindles that form in Eg5-inhibited cells should be resistant to collapse above a certain intercentrosomal threshold distance; in other words, spindles should exhibit an intercentrosomal distance-dependent bistability. To examine this, we utilized a nocodazole washout BAPTA/AM assay [12], which generates mitotic cells containing centrosomes with widely variable positions (in a manner independent of the inhibitors present) (Figure S4). In this assay, LLC-Pk1 cells were treated with nocodazole to completely disassemble MTs and then washed 4X with drug free medium to initiate spindle assembly (see Experimental Procedures). Upon removal of drug, MTs assembled at centrosomes and chromosomes [12]. When centrosomal and chromosomal arrays were close enough to interact (proximal centrosomes), these MT populations quickly coalesced, ultimately resulting in bipolar spindles (9 of 13 cells;Figures 1A and 1B;Table S1;Movies S2andS3); this occurred regardless of the initial spacing between proximal centrosomes. In cells with centrosomal arrays that failed to interact with the chromosomal array (distal centrosomes), acentrosomal bipolar spindles assembled around chromosomes (3 of 5 cells;Figure 1C;Table S1;Movie S4), confirming that mammalian chromosomes alone can organize MTs into bipolar structures.

Yellow lines in B indicate the amputation plane. forms of blindness. In diabetic retinopathy (DR), which affects 150 million patients, neovascularisation of vessels in the adult retina occurs[1]. In age-related macular degeneration (AMD), which is estimated to affect 8 million US patients and 20 million patients in US plus Europe, neovascularisation of capillaries in the choroid layer adjacent to the retina occurs[2]. Ocular neovascularisation associated with retinopathy of prematurity (ROP) arises when premature infants are placed into high oxygen to alleviate respiratory distress. As a consequence, concomitant regression of the lens-associated hyaloid vasculature and growth of the nascent retinal vasculature ceases. When returned to normoxia, the infant retina becomes hypoxic leading to neovascularisation, persistent hyaloid vasculature and eventual blindness[3]. Thus, a therapeutic goal for DR and AMD is to develop agents that inhibit ocular neovascularisation and/or stabilise existing vessels, without diminishing visual function. == Current Ocular Angiogenesis Treatments == Current interventions for resolving inappropriate growth of new vessels in the eye include laser treatment or molecular therapies targeted to vascular endothelial cell growth factor (VEGF). Although the therapeutic mechanism of action is not fully understood, laser photocoagulation surgery can halt neovascularisation and reduce macular oedema[1],[4]. However, success is partial, requires multiple treatments and side effects include cataracts, hemorrhage, retinal detachment and visual field loss. Variations of VEGF molecular therapy to inhibit ocular neovascularisation include antibodies, siRNAs or small molecule inhibitors[5][10]. Those in clinical use are antibodies targeted to VEGF (e.g. bevacizumab (Avastin) and randizumab (Lucentis)) which delay the growth of new 11-hydroxy-sugiol vessels and delay vision loss. Treatment also requires intraocular injection by retinal specialists, repeat administrations and patient sedation. Concerns regarding the development of siRNAs to VEGF have also arisen as they may act non-specifically[7]. More general concerns regarding the strategy of solely targeting VEGF include, the observed compensatory up-regulation of other pro-angiogenic factors which decrease therapeutic efficacy, and the potential to cause retinal neurodegeneration by inhibiting VEGF’s neuroprotectant function[11],[12]. == Alternative Anti Angiogenic Targets == Targeting angiogenic molecules other than, or in addition to VEGF, may reveal more effective and safer inhibitors of intraocular neovascularisation. Potential targets include growth factors (e.g.angiopoietin, FGF, KIR2DL5B antibody HGF, IGF-1, PDGF-B, PlGF), chemokines (e.g.IL8, SDF1, G-CSF), receptors (e.g.CXCR1, FGF-R, PlGFR, PDGFR, Tie-receptors), intracellular mediators (e.g. c-kit kinase, PI3 kinase, PKC) and extracellular mediators (e.g. integrins, cadherins)[13]. Several drugs which do not selectively target VEGF have indeed shown anti-angiogenic efficacy in eyes. 11-hydroxy-sugiol Pazopanib, PKC412 and TG100572 11-hydroxy-sugiol are small molecule inhibitors that each targets multiple kinases[6],[9],[14]. Pazopanib (Armala) which blocks PDGFRs, c-Kit, FGFR and c-fms, and TG100572, which inhibits FGF, PDGF and VEGF, both suppress choroidal neovascularisation in mouse models[6],[9]. TG100572 has improved delivery features as it is effective following local administration as eye drops[6]. PKC412 blocks PKC, VEGF-R, PDGF-R and SCF-R isoforms, and in diabetics is reported to reduce macular oedema[14]. However, liver toxicity was observed following oral administration. Another approach is to selectively target a distinct pro-angiogenic molecule. Indeed recent studies report that the broad-spectrum PI3K inhibitor LY294002 and the v3/v5 integrin antagonist EMD478761 suppress retinal or choroidal neovascularisation following intraocular injection in rodents[15],[16]. A combination of both approaches involves targeting multiple foci of pro-angiogenic pathways with several selective inhibitors. Dorrellet al.report that combination angiostatic therapy with a VEGF aptamer, an integrin antagonist and a proteolytic fragment of tryptophan tRNA synthetase inhibits ocular angiogenesis[11]. == Zebrafish Intraocular Vasculature and Chemical Screens == We recently demonstrated that the inner retina of zebrafish is nourished by an intricate vascular network 11-hydroxy-sugiol which shares many features with human hyaloid and retinal vasculatures[17]. Initially this intraocular vasculature presents as a hyaloid vasculature more tightly associated with the lens which in adults has 11-hydroxy-sugiol migrated to firmly attach to the inner retina. We identified a cohort of genes necessary for normal hyaloid vasculature formation in zebrafish[17]. In addition to amenability towards genetic screens, zebrafish are also suitable for pharmacological screens[18],[19]. In relation to intraocular vasculature, Caoet al.recently show that exposure.