Furthermore, the nucleus could contain more than 1 void (Fig. CENPE: centromere protein E; CIN: chromosomal instability; ConcA: concanamycin A; CQ: chloroquine; DAPI: 4,6-diamidino-2-penylinole; FTI: farnesyltransferase inhibitors; GFP: green fluorescent protein; H2B: histone 2B; KIF: kinesin family member; Light2: lysosomal connected membrane protein 2; MAP1LC3/LC3: microtubule connected protein 1 light chain 3; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; PDS5B: PDS5 cohesin connected element B; SAC: spindle assembly checkpoint; PLEKHM2: pleckstrin homology and RUN domain comprising M2; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; v-ATPase: vacuolar-type H+-translocating ATPase; WAPL: WAPL cohesion launch factor. ((kinesin family member 2?C) [33] or (centromere protein E) [34] produced a 3.3- and 3.8-fold increase of toroidal nucleus population, respectively (Fig. S3I). In all conditions, effectiveness was validated, and lysosomes morphology or distribution were not apparently perturbed (Fig. S3J and S3K). Furthermore, cells treated with nocodazole, a microtubule de-polymerizing agent, showed a 4.5-fold increased prevalence of toroidal nuclei accompanied from the expected lysosomal collapse (Fig. S3I and S3K). We next investigated whether this phenotype is definitely a consequence of impaired nuclear envelope reformation. Live imaging of H2B-GFP U2OS cells stably expressing mCherry-LMNA, indicated the reformation of the nuclear envelope preceded the formation of toroidal nuclei (Fig. S3L and Video 7). This nuclear phenotype is definitely versatile in terms of size, nuclear localization and morphology (Fig. S3M i-iv). Furthermore, the nucleus could contain more than one void (Fig. S3M v, viii) and be accompanied by a micronucleus (Fig. S3M vi). After mitosis, one or both child cells could harbor this phenotype (Fig. S3M vii-viii). We investigated whether toroidal nucleus was a common feature or specific to U2OS cells. To this end, we screened a panel of cell lines. Toroidal nuclei were detected under the tested conditions in more than 80% of cell lines assessed (Number 3L). Among them, non-transformed human pores and skin fibroblast (HFF) and embryonic mouse fibroblasts (MEF), as well as malignancy cells from lung (A549), cervix (HeLa), colon carcinoma (HCT116 and LoVo) or diffuse intrinsic pontine glioma (SU-DIPG-XVII, MC-PED17 and SF-8628) [35C38]. However, we could not detect toroidal nucleus in RKO, MCF7 or HEK293?T cell lines neither in basal population nor less than ConcA or monastrol treatment. Interestingly, the detection Sotrastaurin (AEB071) of toroidal nuclei in all the other cell lines was obvious (compare Physique 3M and S3N), but their sensitivity to induce this nuclear phenotype under the applied treatments varied (Physique 3L). Importantly, most cell lines responded to ConcA-induced blockade of lysosome acidification and to direct mitotic impairment by monastrol as observed by the increased populace of toroidal nuclei (Physique 3L and 3M). Thus, toroidal nuclei emerge as a good read-out for mitotic errors in interphase cells. Lysosome disruption induces the formation of toroidal nuclei The presence of toroidal nuclei in interphase cells facilitates the analysis of mitotic impairment in whole-cell populations, favoring the toroidal nucleus as a powerful tool for quantitative analysis of chromosomal instability. Here, we aimed to screen for lysosome-specific stresses using toroidal nucleus as a biomarker for chromosomal instability. To this end, cells were treated with ConcA or depleted for KIF5B and toroidal nuclei frequency was quantified. Consistently, v-ATPase inhibition as well as blockage of lysosome transport led to a robust increase of toroidal nuclei populace (Physique 4A and S4A). To discard ConcA-side effects not related to v-ATPase inhibition, we genetically or chemically inhibited lysosome acidification. Thus, cells were treated with CQ or depleted of the V0C v-ATPase subunit (silencing RNA and/or with overexpression plasmid. Error bars symbolize S.D. of n ?3 experiments. (G) U2OS cells were depleted for KIF5B or PLEKHM2 and the next day treated or not with ConcA for 24?h. Error bars symbolize S.D of n ?3 experiments. In panels A-B, and D-G nuclei were stained with DAPI for detection and quantification of toroidal nuclei compared to total number of cells. * represents the statistical significance of single treatment effect compared to control, while # represents the effects of the second treatment over control. * p ?0.05, ** p ?0.005, *** p ?0.001, ns: non-significant, # p ?0.05 and ## p ?0.005, ### p ?0.001 Both ConcA and CQ increased toroidal nucleus formation, but their kinetics differed (Fig. S4C). Indeed, ConcA treatment acted faster and produced.The finding that under basal conditions MEF (34831; deposited by Esteban DellAngelica), (34611; deposited by David Sabatini) and (55068; deposited by Michael Davidson). Finally, we characterized an atypical nuclear phenotype, the toroidal nucleus, as a novel biomarker for genotoxic screenings. Our results establish lysosome-dependent degradation as an essential event to prevent chromosomal instability. Abbreviations: 3D: three-dimensional; APC/C: anaphase-promoting complex; ARL8B: ADP ribosylation factor like GTPase 8B; ATG: autophagy-related; BORC: BLOC-one-related complex; CDK: cyclin-dependent kinase; CENPE: centromere protein E; CIN: chromosomal instability; ConcA: concanamycin A; CQ: chloroquine; DAPI: 4,6-diamidino-2-penylinole; FTI: farnesyltransferase inhibitors; GFP: green fluorescent protein; H2B: histone 2B; KIF: kinesin family member; LAMP2: lysosomal associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; PDS5B: PDS5 cohesin associated factor B; SAC: spindle assembly checkpoint; PLEKHM2: pleckstrin homology and RUN domain made up of M2; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; v-ATPase: vacuolar-type H+-translocating ATPase; WAPL: WAPL cohesion release factor. ((kinesin family member 2?C) [33] or (centromere protein E) [34] produced a 3.3- and 3.8-fold increase of toroidal nucleus population, respectively (Fig. S3I). In all conditions, efficiency was validated, and lysosomes morphology or distribution were not apparently perturbed (Fig. S3J and S3K). Furthermore, cells treated with nocodazole, a microtubule de-polymerizing agent, showed a 4.5-fold increased prevalence of toroidal nuclei accompanied by the expected lysosomal collapse (Fig. S3I and S3K). We next investigated whether this phenotype is usually a consequence of impaired nuclear envelope reformation. Live imaging of H2B-GFP U2OS cells stably expressing mCherry-LMNA, indicated that this reformation of the nuclear envelope preceded the formation of toroidal nuclei (Fig. S3L and Video 7). This nuclear phenotype is usually versatile in terms of size, nuclear localization and morphology (Fig. S3M i-iv). Furthermore, the nucleus could contain more than Rabbit Polyclonal to TOP2A one void (Fig. S3M v, viii) and be accompanied by a micronucleus (Fig. S3M vi). After mitosis, one or both child cells could harbor this phenotype (Fig. S3M vii-viii). We investigated whether toroidal nucleus was a common feature or specific to U2OS cells. To this end, we screened a panel of cell lines. Toroidal nuclei were detected under the tested conditions in more than 80% of cell lines assessed (Physique 3L). Among them, non-transformed human skin fibroblast (HFF) and embryonic mouse fibroblasts (MEF), as well as malignancy cells from lung (A549), cervix (HeLa), colon carcinoma (HCT116 and LoVo) or diffuse intrinsic pontine glioma (SU-DIPG-XVII, MC-PED17 and SF-8628) [35C38]. However, we could not detect toroidal nucleus in RKO, MCF7 or HEK293?T cell lines neither in basal population nor under ConcA or monastrol treatment. Interestingly, the detection of toroidal nuclei in all the other cell Sotrastaurin (AEB071) lines was obvious (compare Physique 3M and S3N), but their sensitivity to induce this nuclear phenotype under the applied treatments varied (Physique 3L). Importantly, most cell lines responded to ConcA-induced blockade of lysosome acidification and to direct mitotic impairment by monastrol as observed by the increased populace of toroidal nuclei (Physique 3L and 3M). Thus, toroidal nuclei emerge as a good read-out for mitotic errors in interphase cells. Lysosome disruption induces the formation of toroidal nuclei The presence of toroidal nuclei in interphase cells facilitates the analysis of mitotic impairment in whole-cell populations, favoring the toroidal nucleus as a powerful tool for quantitative analysis of chromosomal instability. Here, we aimed to screen for lysosome-specific stresses using toroidal nucleus as a biomarker for chromosomal instability. To this end, cells were treated with ConcA or depleted for KIF5B and toroidal nuclei frequency was quantified. Consistently, v-ATPase inhibition as well as blockage of lysosome transport led to a robust increase of toroidal nuclei populace (Physique 4A and S4A). To discard ConcA-side effects not related to v-ATPase inhibition, we genetically or chemically inhibited lysosome acidification. Thus, cells were treated with CQ or depleted of the V0C v-ATPase subunit (silencing RNA and/or with overexpression plasmid. Error bars symbolize S.D. of n ?3 experiments. (G) U2OS cells were depleted for KIF5B or PLEKHM2 and the next day treated or not with ConcA for 24?h. Error bars symbolize Sotrastaurin (AEB071) S.D of n ?3 experiments. In panels A-B, and D-G nuclei were stained with DAPI for detection and quantification of toroidal nuclei compared to total number of cells. * represents the statistical significance of single treatment effect compared to control, while # represents the.