Mechanisms of nuclear pore complex disassembly by the mitotic Polo-like kinase 1 (PLK-1) in C. elegans embryos.

The nuclear envelope, which protects and organizes the genome, is dismantled during mitosis. In the Caenorhabditis elegans zygote, nuclear envelope breakdown (NEBD) of the parental pronuclei is spatially and temporally regulated during mitosis to promote the unification of the maternal and paternal genomes. Nuclear pore complex (NPC) disassembly is a decisive step of NEBD, essential for nuclear permeabilization. By combining live imaging, biochemistry, and phosphoproteomics, we show that NPC disassembly is a stepwise process that involves Polo-like kinase 1 (PLK-1)-dependent and -independent steps. PLK-1 targets multiple NPC subcomplexes, including the cytoplasmic filaments, central channel, and inner ring. PLK-1 is recruited to and phosphorylates intrinsically disordered regions (IDRs) of several multivalent linker nucleoporins. Notably, although the phosphosites are not conserved between human and C. elegans nucleoporins, they are located in IDRs in both species. Our results suggest that targeting IDRs of multivalent linker nucleoporins is an evolutionarily conserved driver of NPC disassembly during mitosis.

PLK-1 promotes the merger of the parental genome into a single nucleus by triggering lamina disassembly.

Life of sexually reproducing organisms starts with the fusion of the haploid egg and sperm gametes to form the genome of a new diploid organism. Using the newly fertilized Caenorhabditis elegans zygote, we show that the mitotic Polo-like kinase PLK-1 phosphorylates the lamin LMN-1 to promote timely lamina disassembly and subsequent merging of the parental genomes into a single nucleus after mitosis. Expression of non-phosphorylatable versions of LMN-1, which affect lamina depolymerization during mitosis, is sufficient to prevent the mixing of the parental chromosomes into a single nucleus in daughter cells. Finally, we recapitulate lamina depolymerization by PLK-1 in vitro demonstrating that LMN-1 is a direct PLK-1 target. Our findings indicate that the timely removal of lamin is essential for the merging of parental chromosomes at the beginning of life in C. elegans and possibly also in humans, where a defect in this process might be fatal for embryo development.

Combining near-infrared fluorescence with Brainbow to visualize expression of specific genes within a multicolor context.

Fluorescent proteins are a powerful experimental tool, allowing the visualization of gene expression and cellular behaviors in a variety of systems. Multicolor combinations of fluorescent proteins, such as Brainbow, have expanded the range of possible research questions and are useful for distinguishing and tracking cells. The addition of a separately driven color, however, would allow researchers to report expression of a manipulated gene within the multicolor context to investigate mechanistic effects. A far-red or near-infrared protein could be particularly suitable in this context, as these can be distinguished spectrally from Brainbow. We investigated five far-red/near-infrared proteins in zebrafish: TagRFP657, mCardinal, miRFP670, iRFP670, and mIFP. Our results show that both mCardinal and iRFP670 are useful fluorescent proteins for zebrafish expression. We also introduce a new transgenic zebrafish line that expresses Brainbow under the control of the neuroD promoter. We demonstrate that mCardinal can be used to track the expression of a manipulated bone morphogenetic protein receptor within the Brainbow context. The overlay of near-infrared fluorescence onto a Brainbow background defines a clear strategy for future research questions that aim to manipulate or track the effects of specific genes within a population of cells that are delineated using multicolor approaches.