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The nuclear envelope(NE) isanorganelle ofuniquecomplexand dynamic structure, which serves not only as a physicalbarrier between the nucleus and the cytoplasm, but also as aregulator of many cellular events. During open mitosis, theNE is disassembled in prophase in order to allow the mitoticspindletoaccessthechromosomes[1].NEbreakdownentailsthe disassembly of all the NE components and is associatedwith a series of phosphorylations of proteins of the innernuclear membrane [2], nuclear pore complex [3]andlamina[4], the latest promoting depolymerisation of the lamin fila-ments. The A-type lamins are then solubilised in the cyto-plasm, whereas B-type lamins disperse in the endoplasmicreticulum membranes [5]. Multiple kinases, including cyclin-dependent kinase1(CDK1)anddiacylglycerol-activatedpro-tein kinase Cs (PKCs), have been suggested to play a role inlaminphosphorylationanddisassemblyinvariousmodelsandorganisms,mostlyin cell-free systems [6–17].InCaenorhab-ditiselegans,ahomologueoflipin,alipidphosphatasewhichconverts phosphatidic acid to DAG, is required for lamindisassembly during NE breakdown [18, 19]. To investigatemitotic lamin B1 disassembly, Mall et al. have developed ahigh-throughputlive-imagingassay[20].ThedataobtainedinHeLa cells using this assay confirm previous results suggest-ing the involvement of CDK1, PKCs and lipins in lamindisassembly.This assay is based on the automatic tracking and classi-fication intomitotic phases ofhundredsof individual mitoticcells expressing the chromatin marker H2B and lamin B1chimaeras, monitored by wide-field microscopy. Briefly,nuclei were detected in the H2B-mCherry channel,classified as previously described [21] and the chromatinwas segmented. A ring around the chromatin was defined tomeasure GFP-lamin B1 fluorescence intensity and deducethe assembled or disassembled (in the cytoplasm) state oflamin B1 throughout mitosis. Using a mathematical model,the authors determined the durations of lamin disassemblyin each condition. Mitotic progression from prophase toanaphase and chromosome congression (mean duration ofprometaphase and metaphase) were also calculated.Results obtained with transiently transfected wild-type ormutant lamin B1 reporter using this assay or higher resolu-tion images acquired by confocal microscopy showed thatmutation of the PKC consensus phosphorylation site did notalter lamin disassembly, whereas mutation of the CDK1phosphorylation site alone or combined to the mutation ofthe PKC motif significantly delayed lamin B1 disassemblycompared to controls. However, the differences in the meanduration of lamin disassembly between wild-type andCDK1- or CDK1/PKC-phosphorylation site mutants weresubtle, although significant (7.51±0.87 and 8.24±0.79 minp00.0146 or 8.86±0.80 min, p<0.0001, respectively). Theweakness of the mutants might be attributed to insufficientlevels of expression compared to endogenous proteins(Western blots omitted) and inability to form a network ofmutant proteins which would resist lamina disassembly.Another possibility is that these phosphorylation sites, onlyplay a minor role in lamin disassembly. Relatively highdoses (compared to IC50) of kinase inhibitors, i.e. classicalPKC inhibitor Go6976 and CDK1 inhibitor roscovitinealone or combined,caused significantperturbations of lamindisassembly and mitotic progression, although the twoevents were not correlated, and induced the apparition of‘cytoplasmic fragments’ of lamin B1. Those high intensity‘cytoplasmic fragments’, which were also observed by con-focal microscopy with the CDK1- or CDK1/PKC-sitemutants, could correspond to aggregates of lamin B1 in

Lipid-dependent and -independent regulation of nuclear envelope disassembly